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The endoplasmic reticulum (ER) is a membranousorganelle that plays a crucial role in cell homeostasis. Undercertain conditions, such as those caused by chemical agents,adverse metabolic conditions induce protein misfolding and proteinassembly in the ER, leading to ER stress. Ample evidence implicatesprolonged ER stress in the progression of a number of diseases,including AS (). It has beenconsidered that ER stress is linked to cellular processes withmultiple risk factors in all stages of AS (), and it is believed to play acritical role in cellular apoptosis (). The majority of research on ERstress has focused on the unfolding protein response (UPR), acollection of intracellular signal transduction reactions designedto restore protein homeostasis. In UPR, ER-resident chaperonesprevent ER stress and promote cell survival (). Among these proteins,glucose-regulated protein (GRP)78, a member of the ER heat-shockprotein (HSP)70 family that is the best characterized ER-residentchaperone, is indicative of UPR activation () and is used to determine whether theER stress is activated (). Onthe other hand, some mediators in ER stress have pro-apoptoticeffects; the first of these is the transcriptional activation ofthe gene for C/EBP homologous protein (CHOP), which is ubiquitouslyexpressed at very low levels, but robustly expressed by intensestimulation, ultimately leading to apoptosis.

The THP-1 macrophages were co-incubated withincreasing IVA concentrations (1×10, 2×10,5×10, 1×10 TCID50/ml) as described aboveand collected at 8, 24 and 48 h post-inoculation (pi). The resultsrevealed a pronounced decrease in cell viability in the infectedgroups, as determined by MTT assay (). In early infection (8 h pi),cell viability fluctuated slightly, and the difference was notstatistically significant (P>0.05). The decline in viability wasmore pronounced at 24 h pi. For further confirmation of thisphenomenon, we detected the percentage of apoptotic cells(FITC PI and FITCPI) by flow cytometry at 24 h pi. Similar results wereobserved, except that the number of apoptotic cells followingtreatment with 1×10 TCID50/ml IVA was significantlyhigher compared with the apoptosis observed at the other IVAconcentrations (),suggesting that flow cytometry was more sensitive in detectingapoptotic cells than MTT assay.

Having established that the caspase-dependentmitochondrial pathway was activated in IMQ-induced cell apoptosis(,), we wished to determine whether ERstress also participates in IMQ-induced apoptosis. To examine thishypothesis, GRP78 and CHOP expression was detected by RT-PCR,qRT-PCR and western blot analysis. As shown in , the levels of GRP78 andCHOP mRNA were increased following treatment with IMQ, particularlyat a high concentration (10 μg/ml). The protein levels of GRP78 andCHOP were consistent with the mRNA levels (). These results suggest that ERstress plays a pro-apoptotic role in IMQ treatment, particularlywith a potent stimulation.